Blood flow-related shear stress induces biochemical and physiological changes in vascular endothelial cells (ECs) through membrane-mediated mechanisms. To understand the molecular basis of plasma membrane-mediated mechanotransduction, we propose new engineering analyses and experimental studies of single EC mechanotransduction. Central to our approach is the novel use of multimodal microscopy including DIG, TIRFM, confocal fluorescence imaging, time-resolved fluorescence, and photonic-force microscopy, all on a single platform. This infrastructure provides experimentally-determined inputs to advanced 3-D image processing algorithms, computational fluid dynamics solvers, and finite element (FE) solid mechanics models enabling time-and position-dependent correlations of cell membrane stresses with lipid-mediated signal transduction. To test our hypothesis that shear stress causes membrane stresses which elicit G-protein activation in gel-phase lipid microdomains we propose 3 specific aims (SAs). Under SA 1 we measure 3-D membrane topology, glycocalyx transport, and anisotropic membrane and cytoplasmic viscoelasticity to develop a full 3-D finite element mechanical model of an EC which predicts the shear-induced membrane stress distribution in the apical surface, cell junctions and focal adhesions. Under SA 2 we test the hypothesis that membrane stress concentrations are correlated with measured shear-induced changes in gel-phase lipid mobility and G-protein activation in EC membranes using time-resolved fluorescence spectroscopy of membrane phase-specific lipoid dyes and BODIPY-GTP, a novel fluorescent ligand for activated-G-proteins. Under SA 3 we use a novel continuous flow waveform generator to test whether prevailing shear stress elicits adaptive changes in cytoplasmic and membrane microrheology and membrane signaling. Results will point to new molecular level interventions for vascular dysfunction and provide the basis for intelligent development of novel biomaterials and tissue engineered blood vessels.